Diagnostic method of cirrhosis and hepatic cancer

ABSTRACT

This invention relates to a method of diagnosing cancerous diseases, which comprises measuring the amount of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyl-transferase in body fluid and evaluating the increase in its amount for the diagnosis of hepatic diseases. 
     AFP, CEA and γ-glutamyltranspeptidase have hitherto been used as tumor markers for the diagnosis of hepatic cancer. But these conventional tumor markers show a positivity rate of about 60%, making early diagnosis almost impossible. 
     The method of this invention employs UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase as tumor marker, whereby early diagnosis of hepatic cancer can be made almost completely.

FIELD OF THE INVENTION

This invention relates to a method of diagnosing cancerous diseases.

More particularly, it relates to a method of diagnosing cancerousdiseases of the liver, etc. based on the increase in the amount ofUDP-N-acetyl-glucosamine:glycoprotein N-acetylglucosaminyltransferaseIII (hereinafter abbreviated a Gn-T-III ) in body fluid.

The method of this invention allows simple diagnosis of cancerousdiseases such as hepatic cancer (hepatocirrhosis) by measuring theincrease in the amount of Gn-T-III in body fluid ( e.g., serum, salivaand urine ), and hence will be of much benefit to the medical anddiagnostic fields.

PRIOR ART AND PROBLEMS TO BE SOLVED BY THE INVENTION

GOT, GPT, LDH, ChE and many other test items have been adopted forgeneral diagnosis of hepatic functions.

These test items, however, are no more than to check the comparativedegree of hepatic functions, and are far from direct diagnosis ofhepatic diseases, particularly hepatic cancer.

Measurement of tumor markers, such as AFP and CEA, is also known to benecessary for the diagnosis of hepatic cancer and has been put intopractice.

But these conventional tumor markers show a positivity rate of 60 % atthe highest, making early diagnosis almost impossible.

Recently, γ-glutamyltranspeptidase is receiving attention as a new tumormarker (particularly for hepatic cancer), because of the new fact thatthe blood of patients with hepatic cancer contains glycoproteinscarrying different sugar-chain structure compared with normal subjects.However, this γ-glutamyltranspeptidase is not better than AFP, CEA andothers as a tumor marker.

MEANS TO SOLVE THE PROBLEMS

Detailed studies on the change in sugar-chain structure in patients withhepatic cancer revealed that N-acetylglucosamine is attached, throughβ1,4-linkage, to the mannose (of β-1,4-linkage) bound to the trimannosylcore of sugar chain of asparagine linked type. We continued ourinvestigation on the assumption that this change might be accompanied bythe increase in the amount of Gn-T-III--an enzyme capable oftransferring this N-acetylglucosamine. As a result, it was demonstratedthat the sera of patients suffering hepatic diseases (particularlyhepatic cancer) show a significantly higher Gn-T-III activity comparedwith normal subjects. We then succeeded in establishing a simple methodfor measuring the amount of this enzyme. The present invention wasaccomplished on the basis of these findings.

It was first found by the present inventors that the sera of normalsubJects generally show a Gn-T-III activity as low as about 2.0±0.5nmol/ml/h, while the sera of patients with hepatic cancer have about 2to 3 times the activity, the sera of patients with hepatocirrhosis about1.5 times and the sera of patients with chronic hepatitis 1.2 times.

On page 634 of Preliminary Notes for the 60th Meeting of JapaneseBiochemical Society, is described a method of measuring Gn-T-IIIactivity, in which N-acetylglucosamine is transferred to GnGn sugarchain and the product thus formed is measured by high-performance liquidchromatography. However, it is not known at all to apply this method tothe diagnosis of cancerous diseases.

In the method of this invention, the amount of Gn-T-III is preferablymeasured by allowing it to act upon uridine diphosphoN-acetylglucosamine (hereinafter abbreviated as UDP-GlcNAc) and totransfer N-acetylglucosamine to GnGn sugar chain. Thus the productformed is detected by high-performance liquid chromatography. In thiscase, if the GnGn sugar chain is previously fluorescence-labelled, theproduct can be easily detected by monitoring the fluorescence intensity.The GnGn sugar chain used in this invention is isolated from humantransferrin, and then pyridylaminated (fluorescence labelling) by themethod of Hase et al. (S. Hase et al, Journal of Biochemistry, 197-203(1984), as shown by formula (I). ##STR1##

β-Galactosidase is then allowed to act upon this sugar chain, givingpyridylaminated GnGn sugar chain of formula (II). ##STR2##

The GnGn sugar chain herein means the part of compound (II) from which2-aminopyridine (fluorescent substance) is removed, and it also includesa derivative thereof in which fucose is attached to the 1-position(GlcNAc).

The reaction of Gn-T-III in the method of this invention is shown by thefollowing equation (III): ##STR3##

The reaction mixture was subjected to high-performance liquidchromatography, and the amount of reaction product was determined fromthe fluorescence-intensity, thus measuring the enzyme activity ofGn-T-III.

The amount of Gn-T-III may also be measured by other methods, such as bythe antigen-antibody reaction.

EFFECTS ACHIEVED BY THE INVENTION

It was demonstrated that hepatic disease increases the Gn-T-III activityin the serum, and that this enzyme activity can be easily measured byallowing it to act upon UDP-ClcNAc to transfer N-acetylglucosamine toGnGn sugar chain and determining the amount of reaction product byhigh-performance liquid chromatography. This invention provides a simplemethod for diagnosing cancerous diseases such as hepatic cancer based onthese findings.

Presented below is an Example of this invention.

EXAMPLE

    ______________________________________                                        Reagent                                                                       ______________________________________                                        250 mM    MES (2-(N-morpholino)ethanesulfonic acid                                      monohydrate) (pH: 6.25)                                             400 mM    GlcNAc (N-Acetylglucosamine)                                         20 mM    MnCl.sub.2                                                           40 mM    UDP-GlcNAc                                                          1.0%      Triton X-100                                                        150 μM GnGn sugar chain (flurorescence-labelled)                           ______________________________________                                    

Into fifty containers each containing 50 μl of the above reagent, wereadded 50 μl of sera taken from patients with primary hepatic cancer,patients with hepatocirrhosis, patients with chronic hepatitis, patientswith fatty liver and normal persons (1 cases each), the mixtures wereincubated at 37° C. for one hour, and the reaction was terminated byadding 20 μl each of a solution containing 0.2M EDTA and 0.1M sodiumborate.

Each of the reaction mixtures (1 μl) was subjected to high-performanceliquid chromatography, fluorescene-intensity chromatograms wereprepared, and the Gn-T-III relative activity was determined for eachcase.

The result is shown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                                         Gn-T-III Relative Activity                                   ______________________________________                                        Serum of patients with primary                                                                   3.7 ± 2.3                                               (n mol/n/ml serum)                                                            hepatic cancer                                                                Serum of patients with hepato-                                                                   3.3 ± 1.8                                               cirrhosis                                                                     Serum of patients with chronic                                                                   2.0 ± 0.5                                               hepatitis                                                                     Serum of patients with fatty                                                                     2.0 ± 0.5                                               liver                                                                         Serum of normal persons                                                                          2.0 ± 0.5                                               ______________________________________                                    

What is claimed is:
 1. A method for diagnosing hepatocirrhosis orhepatic cancer which comprises the following steps:(a) addingfluorescence-labelled GnGn sugar chain and UDP-GlcNAc to a serum sampleto react with UDP-N-acetylglucosamine:glycoproteinN-acetylclucosaminyltransferase III (Gn-T-III) in said serum sample toproduce the following compound: ##STR4## (b) subjecting the resultingreaction solution containing said compound to high-performance liquidchromatography; and (c) examining the increase in degree of Gn-T-IIIactivity.